BIOTECHNOLOGY

CATEGORY L T P CREDIT
BTT304 DOWNSTREAM PROCESSING
PCC 3 1 0 4

Preamble: This course is important to understand the basic processes on-going in a

Biological Fermentation from both upstream and downstream processing.

Prerequisite: Basics of biochemical engineering and unit operations

Course Outcomes: After the completion of the course the student will be able to

CO 1 Describe the principles that underlie major unit operations used in downstream
processing of biotechnological and biopharmaceuticals
CO 2 Define and carry out separation and purification of fermentation products
CO 3 Integrate biological and engineering principles involved in the production and
recovery of commercial products.
CO 4 Design and formulate effective strategies of downstream processing based on
characteristics of biomolecules
CO5 Analyse the quality and characteristics of the purified product
CO6 Demonstrate the suitable downstream approaches comprising of new concepts and
emerging technologies

Mapping of course outcomes with program outcomes

PO 1 PO 2 PO 3 PO 4 PO 5 PO 6 PO 7 PO 8 PO 9 PO PO PO
10 11 12
CO 1 - - 3 2 - 2 - - - - - -
CO 2 - - 2 2 - 2 - - - - - -
CO 3 - - 3 2 - 2 - - - - 2 -
CO 4 - - 3 3 3 2 - - - - 2 -
CO5 - - 2 2 - 2 - - - - - -
CO6 - - 3 3 3 2 - - - - - -

Assessment Pattern

Bloom’s Category Continuous Assessment End Semester Examination

Tests
1 2
Remember 10 10 10
Understand 20 20 20
 BIOTECHNOLOGY
Apply 20 20 70
Analyse
Evaluate
Create

Mark distribution

Total CIE ESE ESE Duration

Marks

150 50 100 3 hours

Continuous Internal Evaluation Pattern:

Attendance : 10 marks
Continuous Assessment Test (2 numbers) : 25 marks
Assignment/Quiz/Course project : 15 marks

End Semester Examination Pattern: There will be two parts; Part A and Part B. Part A
contain 10 questions with 2 questions from each module, having 3 marks for each question.
Students should answer all questions. Part B contains 2 questions from each module of
which student should answer any one. Each question can have maximum 2 sub-divisions
and carry 14 marks.

Course Level Assessment Questions

Course Outcome 1 (CO1): Describe the principles that underlie major unit operations used
in downstream processing of biotechnological and biopharmaceuticals

1. Discuss the mechanism of action of a detergent on the cell wall?

2. Show that enzymatic lysis is useful in sequential release of products

3. Discuss the process of Cavitation and its role in DSP

Course Outcome 2 (CO2): Define and carry out separation and purification of fermentation
products

1. Differentiate between HVLV and LVHV products with examples

2. Illustrate the principle and application of foam fractionation

3. Outline the major steps involved in the product isolation and purification of any one
intracellular enzyme
 BIOTECHNOLOGY
Course Outcome 3(CO3): Integrate biological and engineering principles involved in the
production and recovery of commercial products.

1. Justify that “ultracentrifugation is useful in studying Subunit Stoichiometry of

biomolecules?

2. Differentiate between dead and cross flow filtration with neat sketch??

3. Discuss the principle of separation of charged species by IEC

Course Outcome 4 (CO4): Design and formulate effective strategies of downstream

processing based on characteristics of bio molecules

1. Explain working principle of Reverse micellar and supercritical fluid extraction?

2. What are aqueous biphasic systems, give steps involved in the aqueous two phase
extraction of an enzyme

3. Differentiate between dead and cross flow filtration with neat sketch??

Course Outcome 5(CO5): Analyse the quality and characteristics of the purified product

1. Compare gel polarization and fouling. Discuss the factors which contribute to fouling
of membranes.

2. Explain the principle of Isoelectric focussing. Append a neat sketch

3. Differentiate between Perstraction and Pervaporation

Course Outcome 6(CO6): Demonstrate the suitable downstream approaches comprising of

new concepts and emerging technologies

1. Analyse the importance of in-situ bioproduct recovery and bioprocess integration in

downstream processing

2. Explain Mier’ssuper saturation theory of crystallization.

3. With a neat sketch, describe the construction and operation of any one industrial
crystallizer
 BIOTECHNOLOGY
Model Question Paper

Total Pages:
Reg No.:______________ Name:_______________________
APJ ABDUL KALAM TECHNOLOGICAL UNIVERSITY
SIXTH SEMESTER B. TECH DEGREE EXAMINATION ________ ____ 20__
Course Code: BTT 304
Course Name: DOWNSTREAM PROCESSING
Max. Marks: 100 Duration: 3 Hours
PART A
Answer all questions, each carries 3 marks.
1 a) Discuss the electrical double layer concept
b) Discuss the kinetics of bead milling
c) Brief about the advantages and disadvantages of Ultra sonication
d) Elaborate the principle of Reverse micellar extraction theory

e) Differentiate between Orthokinetic and perikinetic aggregation

f) Explain the phenomenon of concentration polarization

g) How the enzymatic lysis is useful in sequential release of products

h) Explain the mechanism of gravity settling

i) Differentiate between HVLV and LVHV products with examples

j) Illustrate the principle and application of foam fractionation

PART B
Answer any one full question from each module. Each carries 14 marks.
2 a) List out the various cell disruption techniques and their mechanism (10)

b) Explain the working principle of a high pressure homogenizer with the help (4)
of a neat sketch
OR
3 a) Differentiate between dead and cross flow filtration with neat sketch?? (10)

b) Discuss the principle of separation of charged species by IEC (4)

4 a) Draw a neat sketch and explain the principle of the following: tubular bowl (8)
centrifuge ,disc stack centrifuges
b) A continuous disc stack centrifuge is operated at 5000 rpm for separation (6)
of bakers’ yeast. At a feed rate of 60 L min−1, 50% of the cells are
recovered. For operation at constant centrifuge speed, solids recovery is
inversely proportional to the flow rate.
 BIOTECHNOLOGY
(a)What flow rate is required to achieve 90% cell recovery if the centrifuge
speed is maintained at 5000 rpm?
(b) What operating speed is required to achieve 90% recovery at a feed
rate of 60 L min−1?
OR
5 a) Differentiate between Perstraction and Pervaporation (8)
b) Analyse the importance of in-situ bio product recovery and bioprocess (6)
integration in downstream processing
6 Outline the principle, operation, merits and limitations of supercritical fluid (14)
chromatography. Discuss its benefits over liquid chromatography.

OR
7 a Explain the principle of Isoelectric focussing. Append a neat sketch (7)
Explain the principle of Bonded phase chromatography with neat sketch (7)
8 a List out various equipment used for conventional filtration and their (8)
working principles
b Describe Aq. Two phase extraction and reverse micellar extraction with (6)
applications
OR
9 a) Explain Mier’ssuper saturation theory of crystallization. (6)

b) With a neat sketch, describe the construction and operation of any one (8)
industrial crystallizer
10 a) Explain the basic instrumentation and working of Liquid chromatography (14)
OR
11 a) List out different types of Commercial dryers with necessary explanation (14)

****

Syllabus

Module 1:

Overview of bio separations: Broad classification of bio products, characteristics of

fermentation broths, Introduction to high volume, low value products and low volume, high
value products, need for downstream processing, criteria for choice of recovery processes,
problems and requirements of bio product purification

Cell disruption: Analysis of various physical, chemical, enzymatic and mechanical methods
for release of intracellular products- kinetics of bead milling and high pressure
homogenization.
 BIOTECHNOLOGY
Module 2:

Flocculation: Importance in downstream processing, electrical double layer concept, DLVO

theory, mechanisms of charge dependent flocculation ,Foam and bubble fractionation:
Principle and operation-applications ,Centrifugal bio separations: Theory of centrifugal
settling- basic equations, mechanism of sedimentation, Sedimentation coefficient,
centrifuge selection-RCF, scale up of centrifuges- sigma analysis, equivalent time- Isopycnic
sedimentation, ultra centrifugation,

Filtration: Equipment for conventional filtration- filter media, pre-treatment methods,

general filtration theory- Darcy’s law, compressible and incompressible filter cakes, filtration
cycle, scale up and design of filtration systems, laboratory filtration tests- batch pre-
treatment test, funnel filtration tests, filter leaf tests.

Module 3:

Extractive bio separations: General principles, analysis of batch and staged extraction -
analytical and graphical methods, scale up and design of extractors- reciprocating plate
extraction columns, centrifugal extractors- aqueous two phase extraction, reversed micellar
extraction and supercritical fluid extraction theoretical principles, process, equipment and
applications.

Precipitation: Factors influencing protein solubility, methods of precipitation, precipitate

formation phenomena orthokinetic and perikinetic aggregation- Smoluchowski’s equation-
precipitate ageing- Camp number- design of precipitation systems.

Module 4:

Membrane separation processes: Cross flow filtration – filter media- ultra filtration and
microfiltration membranes, filter modules, modes of operation, concentration polarization
and fouling-reverse osmosis, dialysis, electro dialysis, Pervaporation, Perstraction.

Chromatographic separations: Classification of techniques, elution chromatography-

retention theory, band broadening effects, separation efficiency, resolution, yield and
purity. Bonded phase chromatography, Ion exchange chromatography, gel permeation
chromatography, affinity chromatography- supercritical fluid chromatography – Chiral
chromatography- expanded bed chromatography- simulated counter current
chromatography- process scale up. Electro kinetic separations: Electrophoresis – Principles
and techniques- Immunoelectrophoresis, capillary zone electrophoresis - Isoelectric
focusing, isotachophoresis
 BIOTECHNOLOGY
Module 5:

Product crystallization: Basic principles- nucleation and crystal growth- Mier’ssuper

saturation theory- kinetics of crystallization-analysis of dilution batch crystallization-
commercial crystallizers- process crystallization of proteins scale up and design of
crystallizers- Recrystallization. Product drying: Heat and mass transfer in drying- types of
commercial dryers- vacuum dryers, freeze dryers, spray dryers- scale up and design of
drying systems. Modern strategies: Bioprocess integration, intensification, in situ bioproduct
recovery, combined operations- whole broth processing, mass recycle

Text Books

1. Sivasankar B, Bio separations: Principles and Techniques, Prentice-Hall of India Pvt. Ltd.,
2008.

2. Paul A Belter, EL Cussler, Wei-shou Hu, Bio separations: Downstream Processing for
Biotechnology - Wiley Interscience, 1988.

Reference Books

1. Harrison RG, Todd P, Rudge SR, Petrides DP, Bio separations Science and Engineering,
Oxford Press, 2003.

2. Richard W Baker, Membrane Technology and applications, John Wiley & Sons Ltd., 2004.

3. McCabe, WL, Smith JC, Harriott P, Unit Operation of Chemical Engineering, 6/e, McGraw
Hill, New York, 2000.

Course Contents and Lecture Schedule

No Topic No. of Lectures

1 Module 1:Overview of bio separations
1.1 Broad classification of bio products, characteristics of 1
fermentation broths
1.2 Introduction to high volume, low value products and low volume, 1
high value products, need for downstream processing
1.3 criteria for choice of recovery processes, problems and 1
requirements of bio product purification
1.4 Cell disruption: Analysis of various physical, chemical, enzymatic 3
and mechanical methods for release of intracellular products
1.5 Kinetics of bead milling and high pressure homogenization. 2
2 Module 2:Flocculation
2.1 Flocculation: Importance in downstream processing, electrical 2
 BIOTECHNOLOGY
double layer concept, DLVO theory, mechanisms of charge
dependent flocculation
2.2 Foam and bubble fractionation: Principle and operation- 1
applications
2.3 Centrifugal bio separations: Theory of centrifugal settling- basic 2
equations, mechanism of sedimentation, Sedimentation
coefficient
2.4 Centrifuge selection-RCF, scale up of centrifuges- sigma analysis, 1
equivalent time-
2.5 Isopycnic sedimentation, ultra centrifugation. 1
2.6 Filtration: Equipments for conventional filtration- filter media, 1
pre-treatment methods, general filtration theory- Darcy’s law,
2.7 Compressible and incompressible filter cakes, filtration cycle, 1
scale up and design of filtration systems,
2.8 Laboratory filtration tests- batch pre-treatment test, funnel 1
filtration tests, filter leaf tests.
3 Module 3:Extractive bioseparations
3.1 General principles, analysis of batch and staged extraction - 1
analytical and graphical methods,
3.2 Scale up and design of extractors- reciprocating plate extraction 1
columns, centrifugal extractors
3.3 Aqueous two phase extraction, reversed micellar extraction and 1
3.4 Supercritical fluid extraction theoretical principles, process, 1
equipment and applications.
3.5 Precipitation: Factors influencing protein solubility, methods of 2
precipitation,

3.6 Precipitate formation phenomena orthokinetic and perikinetic 1

aggregation

3.7 Smoluchowski’s equation-precipitate ageing- Camp number- 1

design of precipitation systems

4 Module 4 :Membrane separation processes:

4.1 Crossflow filtration – filter media- ultra filtration and 1
microfiltration membranes,
4.2 Filter modules, modes of operation, 1
4.3 Concentration polarization and fouling 1
4.4 -reverse osmosis, dialysis, electrodialysis, Pervaporation, 1
Perstraction.
4.5 Chromatographic separations: Classification of techniques, 2
 BIOTECHNOLOGY
elution chromatography- retention theory, band broadening
effects, separation efficiency, resolution, yield and purity.
4.6 Bonded phase chromatography, Ion exchange chromatography, 1
gel permeation chromatography,
4.7 Affinity chromatography- supercritical fluid chromatography – 1
Chiral chromatography-
4.8 Expanded bed chromatography- simulated counter current 1
chromatography- process scale up.
4.9 Electro kinetic separations: Electrophoresis – Principles and 1
techniques- Immuno electrophoresis

4.10 capillary zone electrophoresis - Isoelectric focusing, 1

isotachophoresis

5 Module 5 :Product crystallization:

5.1 Basic principles- nucleation and crystal growth- 1
5.2 Mier’ssupersaturation theory- kinetics of crystallization-analysis 1
of dilution batch crystallization-
5.3 Commercial crystallizers- process crystallization of proteins scale 1
up and design of crystallizers- Recrystallization.
5.4 Product drying: Heat and mass transfer in drying- types of 1
commercial dryers- vacuum dryers, freeze dryers,
5.5 Spray dryers- scale up and design of drying systems. 1
5.6 Modern strategies: Bioprocess integration, intensification, in situ 3
bioproduct recovery, combined operations- whole broth
processing, mass recycle